Tissue and organ transplants save many lives threatened by disease and cancer each year. A particularly medically useful type of transplantation is allogeneic bone marrow transplantation (BMT). Allogeneic bone marrow transplantation may be used to remedy acquired defects in either the hematopoietic system or the immune system, since both types of cells develop from a common stem cell. Furthermore, allogeneic bone marrow transplantation provides a means of correcting inherited enzymatic deficiencies or other genetic defects by providing a self-renewing source of the particular enzyme or other gene product missing in the affected individual.
Still further, allogeneic bone marrow transplantation may be used to treat bone marrow malignancies—i.e. leukemias. Typically, treatment of leukemia involves the use of chemotherapeutic agents which destroy both the patient's normal bone marrow stem cell populations and the leukemia cancer cell populations. Accordingly, allogeneic bone marrow transplantation must be used following high-dose myeloablative chemotherapy and/or radiation therapy to restore the normal red and white blood cell progenitor cell populations in the patient. For the treatment of other cancers not involving the patient's bone marrow, the patient's own bone marrow may be harvested prior to and reinfused following chemotherapy and/or radiation therapy in what is called an autologous bone marrow transplant.
Due to the inability to transfer only the stem cell population, the applicability of allogeneic BMT remains restricted by graft vs. host disease (GVHD), which is apparently mediated mainly by T lymphocytes in the graft cell population. Risk of GVHD has limited allogeneic BMT to use only in highly fatal diseases, and even then, only for patients with HLA-matched donors, usually siblings. Autologous BMT can avoid most of the problems associated with allogeneic transplants. In autologous BMT, however, it is necessary to reintroduce only desirable cell populations free of diseased cell populations (e.g., occult tumor cells) to avoid re-introduction of the disease.
Many of the problems associated with both allogeneic and autologous BMT can be alleviated by using purified stem cell populations for the graft. Purified stem cell populations can be obtained from marrow cell suspensions by positive selection (collecting only the desired cells) or negative selection (removing the undesirable cells), and the technology for capturing specific cells on affinity materials is well developed (Wigzel et al., (1969) J. Exp. Med., 129:23; Schlossman et al., (1973) J. Immunol., 110:313; Mage et al., (1977) J. Immunol. Meth., 15:47; Wysocki et al., (1978) Proc. Nat. Acad. Sci., 75:2844; Schrempf-Decker et al., (1980) J. Immunol. Meth., 32:285; Muller-Sieburg et al., (1986) Cell, 44:653).
Monoclonal antibodies against antigens peculiar to mature, differentiated cells have been used in a variety of “negative” selection strategies to remove undesired cells (i.e. to deplete T cells or malignant cells from allogeneic or autologous marrow grafts respectively) (Gee et al., (1988) J.N.C.I. 80:154-9; Gee et al., (1987) “Proc. of 1st Int. Workshop on Bone Marrow Purging” in Bone Marrow Transpl., Supp. 2, London, MacMillan). Successful purification of human hematopoietic cells by negative selection with monoclonal antibodies and immunomagnetic microspheres has been reported which involved the use of multiple monoclonal antibodies, thus making it more costly for clinical application than positive selection (Griffin et al., (1984) Blood, 63:904; Kannourakis, et al., (1987) Exp. Hematology, 15:1103-1108). Furthermore most studies report only 1 to 2 orders of magnitude reduction in the target cell level following monoclonal antibody treatment. This may not be adequate T lymphocyte depletion necessary to prevent GVHD in allogeneic transplants, and it is certainly insufficient for the purpose of removing cancer cells in autologous bone marrow transplantation where 106 to 109 malignant cells may be present in the patient's marrow.
Positive selection of normal marrow stem cells is an alternative for treatment of the bone marrow graft. The procedure employs a monoclonal antibody which selectively recognizes human lymphohematopoietic progenitor cells, such as the anti-MY10 monoclonal antibody that recognizes an epitope on the CD34 glycoprotein antigen. Cells expressing the CD34 antigen include essentially all unipotent and multipotent human hematopoietic colony-forming cells (including the pre-colony forming units (pre-CFU) and the colony forming unit-blasts (CFU-Blast)) as well as the very earliest stage of committed B lymphoid cells, but NOT mature B cells, T cells, NK cells, monocytes, granulocytes, platelets, or erythrocytes. See Civin, U.S. Pat. No. 4,714,680. This method of isolating CD34+ cells results in a mixed cell population of stem and progenitor cells that includes all lineages and stages of lympho-hematopoietic stem and progenitor cells and some later precursor cells. Such positive selection procedures additionally suffer from some disadvantages including the presence of materials such as antibodies and/or magnetic beads on the CD34+ cells, and damage to the cells resulting from the removal of these materials. In addition, researchers want to focus down on only the most primitive of the cells within the CD34+ cell population (see below).
Accordingly, there is a continued interest in finding other methods to either replace or augment current methods of isolating cell populations that are enriched in primitive in vivo engrafting hematopoietic stem cells. One way to achieve this is to gain a better understanding of the molecular signature of in vivo engrafting hematopoietic stem cells and on this basis, develop better methods of obtaining purer populations of such stem cells.
The study of hematopoiesis until recently, has been limited because of the complexity of isolating a homogenous purified stem cell population. A small number of in vivo engrafling (lympho-)hematopoietic stem cells (HSCs), present in bone marrow (BM), placental/umbilical cord blood (CB), or growth-factor-mobilized peripheral blood (PBSC) give rise to progressively more lineage-committed hematopoietic progenitor cells (HPCs), which in turn produce all of the mature blood and immune cells, and probably endothelial cells as well. In humans, most HSCs and HPCs express the CD34 phosphoglycoprotein protein and MRNA. In vivo engrafling HSCs comprise <<1% of the total CD34+ cell population. Other markers, such as efficient efflux pumping of rhodamine or Hoescht dyes, or CD133, that enrich for primitive hematopoietic stem-progenitor cell (HSPC) subpopulations have also been described, but are much less extensively characterized for human as opposed to mouse HSPCs, with regard to HSC function such as repopulation and engraftment ability (Civin et al., (1996) J Clin Oncol., 14:2224-2233; Larochelle et al., (1996) Nat Med., 2:1329-1337; Krause et al., (1996) Blood, 87:1-13; Civin et al., (1984) J. Immunology, 133:157-165 and Bhatia et al., (1998) Nat Med., 4:1038-45).
A significant body of work has been reported on the gene expression of mouse HSPCs. For example, initial studies used cDNA/RT-PCR-based subtraction libraries of transcripts expressed in mouse fetal liver (Phillips et al., (2000) Science, 288:1635-1640) or BM (Terskikh et al., (2001) Proc. Natl. Acad. Sci. U.S.A., 98:7934-7939) HSPCs, and found hundreds to thousands of transcripts over-represented in HSPCs, as compared to more mature hematopoietic cells. Park et al., ((2002) Blood, 99:488-498), using a subtractive microarray approach to compare mouse HSC-enriched Thy1.1loc-kit+Sca−1hiLin−/lo cells to HPC-enriched populations, found that approximately 5000 cDNA clones were differentially expressed between the two populations. Terskikh et al., ((2001) Proc. Natl. Acad. Sci. U.S.A., 98:7934-7939) used nylon cDNA arrays, containing a limited set of 1,176 genes, to examine gene expression of mouse HSCs, common myeloid, granulocyte-macrophage, megakaryocyte-erythrocyte, and lymphoid progenitors, and pro-B, and pro-T cells. Although this study examined only a handful of genes, the authors showed that a number of hematopoiesis-specific genes were expressed by HSCs. The expression of these genes decreased in progressively more committed HPCs, which at the same time, began to express lineage-specific genes. Akashi et al., ((2003) Blood, 101:383-389 ) performed a similar study with 24,000 gene oligonucleotide arrays. In addition to confirming the prior study, they found that HSCs expressed a number of “non-hematopoietic” genes.
However, due to the difficulties of isolating numbers of highly purified HSC-enriched sub-populations sufficient to produce the quantities of RNA needed for microarray hybridization, to date only a handful of studies have attempted similar gene expression analyses with human HSPCs. Instead, most previous microarray analyses of human HSPCs have had to use relatively unpurified, “total” CD34+ cell preparations (only <<1% of which are HSCs), rather than more highly HSC-enriched subpopulations of CD34+cells. As an example, Steidl et al., ((2002) Blood, 99:2037-2044) examined the expression of 1185 genes from BM and PBSC (total) CD34+ cells. They found 65 genes differentially expressed, some of which may explain the higher levels of cell cycling in CD34+ cells from BM, as compared to PBSC. A further example includes a recent investigation that analyzed the total CD34+ cell population by SAGE (Zhou et al. (2001) Proc. Natl. Acad. Sci., 98:13966-13971); myeloperoxidase was one of the genes found to be expressed in total CD34+ cells. However, myeloperoxidase is expressed only in committed phagocytic precursors and phagocytes, not in undifferentiated HSCs. (Wang et al., (2001) Leukemia 17:779-786; Friedman et al., (1996) Curr Top Microbiol Immunol., 211:149-157; Friedman et al., (1996) Leuk Res., 20:809-815)
While these studies defined genes expressed in the total CD34+ cell population, these analyses may have missed expression of key human HSC genes or misinterpreted their expression in HSCs versus more mature HPCs. In other words, these studies most likely identified genes expressed principally in HPCs, not HSCs. In addition, only relatively small-scale microarray gene expression analyses have been reported (generally <5000-12,000 known genes), further limiting the impact of these studies of human HSPCs.
Two recent studies have begun to define a general gene expression phenotype for stem cells. Ramalho-Santos et al., ((2002) Science, 298:597-600) examined the transcriptomes of “side population” (SP) mouse BM Kit+Lin−Sca−1+HSC-enriched cells, mouse neurospheres, and a mouse embryonic stem cell (ESC) line. Four transcripts were expressed in all three stem cell types, but not in more mature cell types. An additional 212 transcripts were highly enriched in the three types of stem cells, but these genes were also detected in more mature cell types. Ivanova et al., ((2002) Science, 298:601-4) examined the transcriptomes of mouse adult BM Kit+Lin−Sca−1+ Rholow, mouse fetal liver Kit+Lin−Sca-1+ AA4.1+, and human fetal liver CD34+/CD38−/Lin− HSC-enriched cell populations, as well as mouse neurosphere SP cells and a mouse ESC line. 322 transcripts were enriched in all these HSPC populations, and 283 transcripts in all three stem cell types. Interestingly, both these groups found that approximately half of the genes expressed in the stem cell-enriched populations had unknown function or were ESTs. Yet, similar to previous work with HSPCs, these investigations studied mainly mouse cells, examining only one human cell population. In addition, comparison of the lists of stem cell-overexpressed genes from these two studies reveals that only 6 genes were common to both lists (Fortunel et al., (2003) Science, 302:393; Evsikov et al., (2003) Science, 302:393 and Vogel, G. (2003) Science, 302:393).
Accordingly there is still a need for a detailed molecular characterization of highly enriched human hematopoietic stem cells (HSCs) to identify a set of genes that might include candidate regulators involved in the survival, self-renewal, differentiation and/or migration/adhesion capacities of human HSCs, as well as, genes that may be targets in “cancer stem cells” which give rise to blood cancers.